This application is a xc2xa7371 application of PCT/GB96/03256, filed Dec. 27, 1996.
The present invention relates to a method of measuring cell membrane permeability and is applicable to all types of cells, including red cells, white cells, platelets, fibroblasts, tissue cells, amoebae, fungi, bacteria, all eucaryotic and procaryotic cells as well as synthesized cells or particles.
Permeability is the passage of matter in a fluid or gaseous state through another material, usually in a solid state, measured as a rate or total volume transferred across a membrane per unit time per unit surface area at standard temperature and pressure. Biologically, many membranes, especially cell membranes, are selectively permeable enabling cells to transfer nutrients, hormones, gases, sugars, proteins or water across their membranes. This transport may be passive, depending solely upon the partial pressures or concentrations of the substances on either side of the membrane or it may be active, requiring energy to counter existing concentrations. Different cells have different molecule specific rates of permeability which are closely related to the cell""s function.
Current tests of red cell permeability produce a single value for permeability, typically by measuring the change in concentration of a radio labelled molecule (often water) in or around a cell (or a population of cells).
According to the present invention there is provided a new method in which a sample of cells suspended in a liquid medium, wherein the cells have at least one measurable property distinct from that of the liquid medium, is subjected to analysis to determine a measure of cell permeability of the sample of cells by a method including the steps:
(a) passing a first aliquot of the sample cell suspension through a sensor,
(b) measuring said at least one property of the cell suspension,
(c) recording the measurement of said property for the first aliquot of cells,
(d) subjecting a second aliquot of the sample cell suspension to an alteration in at least one parameter of the cell environment which has the potential to induce a flow of fluid across the cell membranes and thereby alter the said at least one property of the cells,
(e) passing said second aliquot through a sensor,
(f) measuring said at least one property of the cell suspension under the altered environment,
(g) recording the measurement of said at least one property for the second aliquot of cells,
(h) comparing the data from steps (c) and (g) as a function of the extent of said alteration of said parameter of the cell environment and change in the recorded measurements of said at least one property to determine a measure of cell permeability of the sample.
Blood cells travel through the entire body once a minute continually transporting gases and metabolites. Blood cells also act as messengers or surrogate hormones, transmitting information around the body. It has been discovered that this peripatetic existence allows the blood cells to signal distant pathology. For example, when the brain dies, when a limb has an occluded blood supply or the kidney fails to remove essential toxins, the blood cell""s membrane permeability changes. Cell membrane permeability has never been measured routinely and only rarely measured experimentally. Until now, there have been no rapid or reliable methods of performing such measurements. It has also been discovered that red cell permeability is complex, dynamically changing as molecules cross the cell""s membrane depending on, for example, the shape and structure of the cell and membrane pump activity. The method of the present invention produces existing measures of permeability, but more usefully it produces more sensitive, accurate and descriptive measures of cell permeability within sixty seconds with no sample preparation.
Preferably, the property of the cells which differs from the liquid medium is one which is directly related to the volume of the cell. Such a property is electrical resistance or impedance which may be measured using conventional particle counters such as the commercially available instrument sold under the trade name Coulter Counter by Coulter Instruments Inc. Preferably, the sensor used to detect cells and measure a change in the cells"" property is that described in our co-pending International application (Agent""s reference 62/2681/03). In this apparatus the cell suspension is caused to flow through an aperture where it distorts an electrical field. The response of the electrical field to the passage of the cells is recorded as a series of voltage pulses, the amplitude of each pulse being proportional to cell size.
In the preferred method of the present invention, a measurement of cell permeability is determined by obtaining a measure of the volume of fluid which crosses a sample cell membrane in response to an altered environment. The environmental parameter which is changed in the method may be any change which results in a measurable property of the cells being altered. Preferably, a lytic agent is used to drive fluid across the cell membranes and thereby cause a change in cell volume. Preferably therefore, the environmental parameter change is an alteration in osmolality, most preferably a reduction in osmolality. Typically, the environment of the first aliquot is isotonic and thus the environment of the second aliquot is rendered hypotonic. Other suitable lytic agents include soap, alcohols, poisons, salts, and an applied shear stress.
It is possible to subject only a single aliguot of sample suspension to one or more alterations in osmolality to achieve this effect, although is preferred to use two or more different aliquots of the same sample suspension. Most preferably, the sample suspension is subjected to a continuous osmotic gradient, and in particular an osmotic gradient generated in accordance with the method of our co-pending International application (Agent""s reference 80/4936/03).
In the preferred method of our co-pending International application (Agent""s reference 62/2734/03), a number of measurements of particular cell parameters are made over a continuous series of osmolalities, including cell volume and cell surface area, which takes account of the deviation of the cells from spherical shape particles commonly used to calibrate the instruments. An estimate of in vivo cell shape made so that an accurate measurement of cell volume and cell surface area at all shapes is obtained. A sample suspension is fed continuously into a solution the osmolality of which is changed continuously to produce a continuous concentration gradient. Reducing the osmolality of the solution surrounding a red blood cell below a critical level causes the cell first to swell, then rupture, forming a ghost cell which slowly releases its contents, almost entirely haemoglobin, into the surrounding medium. The surface area of the each cell remains virtually unchanged on an increase in cell volume due to a reduction in osmolality of the cell""s environment as the cell membrane is substantially inelastic. The time between initiation of the alteration of the environment in each aliquot to the passage of the cells through the sensing zone is kept constant so that time is not a factor in any calculation in cell permeability. An effect of feeding the sample under test into a continuously changing osmolality gradient, is to obtain measurements which are equivalent to treating one particular cell sample with that continuously changing gradient.
Preferably, the measurements are recorded on a cell-by-cell basis in accordance with the method of our co-pending International application (Agent""s reference 62/2734/03). The number of blood cells within each aliquot which are counted is typically at least 1000 and the cell-by-cell data is then used to produce an exact frequency distribution of cell permeability. Suitably this density can be displayed more visibly by using different colours to give a three dimensional effect, similar to that seen in radar rainfall pictures used in weather forecasting. Alternatively, for a single solution of any tonicity, the measured parameter change could be displayed against a number of individual cells showing the same change. In this way a distribution of cell permeability in a tonicity of given osmolality can be obtained.
As discussed above, the methods in our co-pending applications can provide an accurate estimate of cell volume, or other cell parameter related to cell volume, and cell surface area over a continuous osmotic gradient for individual cells in a sample. A plot of change in cell volume against osmolality reveals a characteristic curve showing how the cell volume changes with decreasing osmolality and indicates maximum and minimum rates of flow across the membrane and the flow rates attributed to a particular or series of osmotic pressures.
Having obtained measures of osmotic pressure (Posm) cell volume, surface area (SA) and other relevant environmental factors, it is possible to obtain a number of measures of cell permeability:
This coefficient of permeability measures the rate of fluid flow across a square meter of membrane in response to a specified pressure. All positive rates represent a net flow into the cell, while all negative rates are the equivalent of a net flow out of the cell. The rate is determined by:
Cp rate=xcex94cell volume/xcex94Posm/SA at S.T.P.
This set of permeability measures describe each pressure where the net permeability rate is zero, and are numbered pk0, pk1 . . . pkn.
(i) pk0 coincides with the minimum absolute pressure (hypotonic) to which a cell can be subjected without loss of integrity. A pressure change of one tenth of a milliosmole per kg (0.0001 atms) at pk0 produces a change in permeability of between one and two orders of magnitude making pk0 a distinct, highly reproducible measure.
(ii) pk1 is a measure of the cells"" ability to volumetrically regulate in slightly hypotonic pressures. After a certain pressure, the cell can no longer defeat the osmotic force, resulting in a change in the cell""s volume. pk1 provides a measure of the cells ability to perform this regulation, thereby measuring a cell""s maximum pump transfer capability.
(iii) pk2, a corollary of pk1, is a measure of the cells ability to volumetrically regulate in hypertonic pressures, and occurs at low differential pressures, when compared to the cell""s typical in vivo hydrostatic pressure.
The permeability constant pkn is described by the following equation:
pkn=xcex94Posm/SA at S.T.P.
When calculating pk0, xcex94Posm=(isotonic pressure)xe2x88x92(pressure where net flow is zero).
When calculating pk1, xcex94Posm=(isotonic pressure)xe2x88x92(first hypotonic pressure where net positive flow begins). The calculation of pk2 is identical to pk1, except xcex94Posm measures the first hypertonic pressure where net positive flow is not zero.
This dimensionless value is the comparison of any two Cp rates, and is expressed as the net amount of fluid to cross the cell membrane between any two lytic concentrations. It provides a volume independent and pressure dependent comparison of permeability rates. This measure may be used to compare permeability changes in the same individual over a period ranging from minutes to months.
This is the maximum rate of flow across the cell""s membrane. For almost all cells, there are two maxima, one positive (net flow into the cell) and one negative (net flow out of the cell) situated either side of ph0. Cpmax is determined by detecting the maximum positive and negative gradients of the continuous curve of change in cell volume against osmolality.
This is a measure of the structural forces inside a cell which resist the in-flow or out-flow of water. It is determined by the ratio of Cpmax to all other non-zero flow rates into the cell. As the membrane is theoretically equally permeable at all pressures, change from the maximum flow rate outside the pressure range of pk1 to pk2 are due to mechanical forces. It is clear that pk0 is an entirely mechanical limit on the cell because as Cprate approaches zero, MSR approaches ∞, thereby producing more strain than the membrane can tolerate.
MSR=Cpmax/Cpratexc3x97100%
This is a measure of the physiological permeability available to an individual per unit volume of tissue or blood, or for the whole organ or total body, and is calculated by:
CP ml=xcex94cell volume/xcex94Posm/m3 per ml of whole blood.
The method of the present invention has a wide range of uses, in particular:
1. A means of measuring permeability and permeability rates on any type of cell.
2. A means for detecting and differentiating normal and abnormal membrane permeabilities and their causes.
3. An in vitro substitute for in vivo animal tests or human experimentation on new drugs, or toxicology experiments, and in particular the effect from unknown substances upon membrane permeability, such as nerve agents, anaesthetics, drugs, radiation and chemical warfare agents.
4. Membrane research.
5. Taxonomy. Different species have different membrane permeabilities which has been known but never used as a basis for taxonomy.
6. A model for other cells, particularly nerve cells, which are dependent upon membrane pumps for nerve impulse propagation.
7. In medicine for blood banking. Currently donated blood units have their shelf life limited to three weeks because some donated blood units do not survive in storage longer than this. However, the majority of units are viable for many more weeks but hospitals do not risk using a non-viable unit for transfusion. The permeability measurements of the present invention provide a means of determining the viability of blood, enabling a quick and cheap method of determining if a unit has expired. It can also be used as a basis for deciding when to discard a unit before the three week limit, thereby reducing the risk of a bad transfusion and potentially saving millions of units each year.
8. As a means for the detection of disease, diagnosis of disease, confirmation of diagnosis, monitoring prognosis of disease, monitoring treatment efficacy and monitoring remission in humans and all other species.
9. As a means of investigating pathophysiology in all species. There are many diseases that have been found to have altered cell membrane permeability that were previously unknown. For example it is altered when insulin binding to the red cell is increased as in anorexia nervosa, when anoxia induced by respiratory failure or congenital diaphragmatic hernia, or in thalassaemia intermedia, due to an undetermined mechanism. Hitherto cell permeability has never been used to monitor blood flow to a limb. One new and unexpected discovery is that occlusion of the blood flow to the lower limb sufficient to require femoral artery bypass, invariably and profoundly changes the red cell membrane permeability.
10. As a means for detecting and confirming death. At death, there is an alteration of cell membrane permeability that is quicker and cheaper to measure than an EEC.
11. Screening of routine samples for abnormality as an indication of disease.